scholarly journals Unmarked gene editing in Clavibacter michiganensis using CRISPR/Cas9 and 5-fluorocytosine counterselection

2021 ◽  
Author(s):  
Xing Chen ◽  
Qing Tan ◽  
Qingyang Lyu ◽  
Chengxuan Yu ◽  
Na Jiang ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Editing ◽  
Gene Knockout ◽  
Resistant Mutant ◽  
Economic Loss ◽  
Site Directed Mutagenesis ◽  
Clavibacter Michiganensis ◽  
Transformation Vector ◽  
Quarantine Species ◽  
Gene Functional Analysis

Plant pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis (Cm), which couples the expression of cas9 and sgRNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the Cm rpsL gene to generate a streptomycin resistant mutant, and complete gene knockout, in which the deletion of the Cm celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 100 μg/ml 5-fluorocytosine (5-FC), in which an unmarked katA mutant lacking the transformation vector was recovered. Compared to conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient as it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into Cm and other Clavibacter species.

scholarly journals Precise gene editing paves the way for derivation ofMannheimia haemolyticaleukotoxin-resistant cattle

2016 ◽  
Vol 113 (46) ◽  
pp. 13186-13190 ◽  
Author(s):  
Sudarvili Shanthalingam ◽  
Ahmed Tibary ◽  
Jonathan E. Beever ◽  
Poothapillai Kasinathan ◽  
Wendy C. Brown ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Gene Editing ◽  
Amino Acid Position ◽  
Economic Loss ◽  
Site Directed Mutagenesis ◽  
Signal Peptidase ◽  
Target Cells ◽  
Cattle Industry ◽  
Peptide Cleavage ◽  
The Us

Signal peptides of membrane proteins are cleaved by signal peptidase once the nascent proteins reach the endoplasmic reticulum. Previously, we reported that, contrary to the paradigm, the signal peptide of ruminant CD18, the β subunit of β2integrins, is not cleaved and hence remains intact on mature CD18 molecules expressed on the surface of ruminant leukocytes. Leukotoxin secreted byMannheimia(Pasteurella)haemolyticabinds to the intact signal peptide and causes cytolysis of ruminant leukocytes, resulting in acute inflammation and lung tissue damage. We also demonstrated that site-directed mutagenesis leading to substitution of cleavage-inhibiting glutamine (Q), at amino acid position 5 upstream of the signal peptide cleavage site, with cleavage-inducing glycine (G) results in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of target cells. In this proof-of-principle study, we used precise gene editing to induce Q(‒5)G substitution in both alleles of CD18 in bovine fetal fibroblast cells. The gene-edited fibroblasts were used for somatic nuclear transfer and cloning to produce a bovine fetus homozygous for the Q(‒5)G substitution. The leukocyte population of this engineered ruminant expressed CD18 without the signal peptide. More importantly, these leukocytes were absolutely resistant to leukotoxin-induced cytolysis. This report demonstrates the feasibility of developing lines of cattle genetically resistant toM. haemolytica-caused pneumonia, which inflicts an economic loss of over $1 billion to the US cattle industry alone.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

scholarly journals Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

2019 ◽  
Vol 48 (2) ◽  
pp. 517-532 ◽  
Author(s):  
Bin Liu ◽  
Siwei Chen ◽  
Anouk La Rose ◽  
Deng Chen ◽  
Fangyuan Cao ◽  
...  
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Rapid Development ◽  
Open Chromatin ◽  
Chromatin Compaction ◽  
Histone Deacetylase 1 ◽  
Cas9 Protein ◽  
Target Dna ◽  
Histone Modifiers ◽  
Low Efficiency

Abstract Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

scholarly journals One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

Cell Research ◽  
2017 ◽  
Vol 27 (7) ◽  
pp. 933-945 ◽  
Author(s):  
Erwei Zuo ◽  
Yi-Jun Cai ◽  
Kui Li ◽  
Yu Wei ◽  
Bang-An Wang ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Gene Editing ◽  
Gene Knockout ◽  
Gene Knockout Mice ◽  
One Step ◽  
Step Generation

scholarly journals Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells

2018 ◽  
Vol 215 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Akiko Seki ◽  
Sascha Rutz
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Editing ◽  
Target Gene ◽  
Gene Knockout ◽  
Great Promise ◽  
Next Generation ◽  
Primary Mouse

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

scholarly journals PDTM-26. NOVEL SHARED AND DISTINCT EPIGENOMIC MECHANISMS OF G34R AND K27M MUTATIONS IN CHILDHOOD GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi192-vi193
Author(s):  
Michael Chen ◽  
Kelly Bush ◽  
Nichole Lewis ◽  
Vanessa Cervantes ◽  
Paul Knoepfler
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Point Mutations ◽  
Glioma Cells ◽  
Driver Mutations ◽  
High Grade ◽  
Shared Key ◽  
Induced Changes ◽  
Open Question

Abstract Alterations in histone H3.3 are common driver mutations in high-grade pediatric gliomas, but the central oncogenic mechanisms remain an open question. To identify important mutant H3.3 effectors, we used CRISPR-Cas9 to precisely introduce H3.3 K27M and G34R mutations into previously H3.3-wildtype human astrocyte and glioma cells, while in parallel reverting mutations in glioma cells back to wildtype. K27M and G34R mutations invoked some strikingly similar epigenomic effects supporting a new model in which some major aspects of their oncogenic functions are shared. For instance, both K27M and G34R induced changes at many of the same genomic loci in specific histone marks, with the largest changes in H3K27me3 including in particular within super-enhancers, which also exhibited perturbed transcriptional function. K27M and G34R mutations induced some gene expression changes that were unique to each mutation, but both mutations changed similar functional ontological clusters and ASCL1 is a shared key putative effector. H3.3 mutant glioma cells are sensitive to ASCL1 knockdown or overexpression, resulting in cell viability that is reduced or increased, respectively. Comparison of our panel of glioma cells gene-edited with precise point mutations to edited glioma cells in other studies that performed gene knockout or overexpression reveals striking differences in the resulting phenotypes. We also determined that certain drugs exhibited specificity to H3.3 mutation-bearing cells including DAPT, JQ1, and ONC201. In vivo, we found that reversion of K27M to WT in glioma cells significantly reduced tumorigenicity in mouse xenograft assays and introduction of G34R mutations in previously WT glioma cells increased tumor growth. Overall, gene editing of gliomas and comparison of otherwise isogenic sets of cells defines both distinct and shared gliomagenesis mechanisms that can be targeted for development of oncohistone-based therapeutics.

scholarly journals Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis

2012 ◽  
Vol 78 (23) ◽  
pp. 8388-8402 ◽  
Author(s):  
Marie-Agnès Jacques ◽  
Karine Durand ◽  
Geoffrey Orgeur ◽  
Samuel Balidas ◽  
Céline Fricot ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Relatedness ◽  
False Negative ◽  
Seed Transmission ◽  
16S Rrna Gene Sequencing ◽  
Housekeeping Genes ◽  
Diagnostic Tools ◽  
Rrna Gene ◽  
Clavibacter Michiganensis ◽  
Content Type

ABSTRACTThe genusClavibactercomprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose.Clavibacter michiganensissubsp.michiganensisis one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection ofC. michiganensissubsp.michiganensisstrains, relatives (strains from the four otherC. michiganensissubspecies), and nonpathogenicClavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD,dnaK,gyrB,ppK,recA, andrpoB). We compared this “framework” with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the mainC. michiganensissubsp.michiganensispathogenicity determinants. We showed thatC. michiganensissubsp.michiganensisis monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenicClavibacter-like strains were identified asC. michiganensisusing 16S rRNA gene sequencing. These strains, while cross-reacting withC. michiganensissubsp.michiganensisidentification tools, are phylogenetically distinct from the pathogenic strains but belong to theC. michiganensisclade.C. michiganensissubsp.michiganensisclonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced.

Gene editing in tomatoes

2017 ◽  
Vol 1 (2) ◽  
pp. 183-191 ◽  
Author(s):  
Joyce Van Eck
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Site Directed Mutagenesis ◽  
Dna Uptake ◽  
Food Crop ◽  
Transcription Activator ◽  
Base Editing ◽  
Crop Models ◽  
Functional Studies ◽  
Tomato Transformation

Tomato is an effective model plant species because it possesses the qualities necessary for genetic and functional studies, but is also a food crop making what is learned more translatable for crop improvement when compared with other non-food crop models. The availability of genome sequences for many genotypes and amenability to transformation methodologies (Agrobacterium-mediated, direct DNA uptake via protoplasts, biolistics) make tomato the perfect platform to study the application of gene-editing technologies. This review includes information related to tomato transformation methodology, one of the necessary requirements for gene editing, along with the status of site-directed mutagenesis by TALENs (transcription activator-like effector nucleases) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated Proteins). In addition to the reports on proof-of-concept experiments to demonstrate the feasibility of gene editing in tomato, there are many reports that show the power of these technologies for modification of traits, such as fruit characteristics (ripening, size, and parthenocarpy), pathogen susceptibility, architecture (plant and inflorescence), and metabolic engineering. Also highlighted in this review are reports on the application of a recent CRISPR technology called base editing that allows the modification of one base pair in a gene sequence and a strategy that takes advantage of a geminivirus replicon for delivery of DNA repair template.

scholarly journals Antimicrobial and genetic strategies to defend kiwifruit against the bacterial pathogen Pseudomonas syringae pathovar actinidiae (Psa)

2021 ◽  
Author(s):  
◽  
Mathew Ambrose Storey
Keyword(s):  
New Zealand ◽  
Pseudomonas Syringae ◽  
Best Practice ◽  
Gene Knockout ◽  
Virulent Strain ◽  
Antimicrobial Properties ◽  
Bacterial Pathogen ◽  
Resistant Mutant ◽  
Potential Biocontrol Agent ◽  
Near Future

<p>A highly virulent strain of the bacterium Pseudomonas syringae pv. actinidiae (Psa-V), the causative agent of bacterial canker of kiwifruit, is threatening the $1.5 billion New Zealand kiwifruit industry. A strain of Psa-V was first identified in Italy in 2008 and related strain with a similar level of virulence arrived in New Zealand in November 2010. Since then it has been spreading rapidly throughout the country with devastating effects. Currently there is no effective treatment for growers to control Psa-V in their orchards and the potential impact of Psa-V on the New Zealand kiwifruit industry and growers is catastrophic. As part of a collaboration between Seeka Kiwifruit Industries, EastPack NZ, and a group of scientists nationwide (Taskforce Green) this work designed and implemented laboratory tests to quantify the effect of candidate antimicrobial sprays. Novel formulations with strong antimicrobial properties, including silver nanoparticles were also tested. This work was complemented by an investigation into the antibiotic resistance potential of Psa-V. A spontaneous streptomycin resistant mutant of Psa-V was generated and the molecular mechanism of resistance was elucidated. Further, gene knockout strategies aimed at facilitating the study of Psa-V virulence genes and ultimately developing a potential biocontrol agent were tested. Overall, this work together with several recent advances in the field should help advise the kiwifruit industry on best practice around the use of anti-Psa-V agents, and may make it possible to facilitate the generation and testing of candidate biocontrol agents in the near future.</p>

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